Some of the projects carried over and continued from the previous year but new collaborations were also initiated with several intramural researchers during this year. We continued our NIDCR (Yamada Lab) collaboration but attention was turned to the effect of substrate mechanical properties on cell motility and other cell characteristic behavior. This time our collaborators investigated the behavior of cells on substrates with variable stiffnesses. They were preparing substrate gels with tailored elastic modulus gradients and followed the movement of cells relative to the gradients. For each substrate we performed extensive measurements to establish the protocol for their preparation. These substrates were polyacrylamide gels coated with typical, physiological components of extracellular matrix (ECM) such as collagen, fibronectin or laminin. These coatings have to be finely tuned to allow the cells to feel the underlying stiffness gradients. In addition, we have been investigating various protocols for preparing gradients that do not require the use of polymers like acrylamide (not a physiological component of ECM) but rather use dominant components of the ECM directly. One such approach employs magnetic microspheres that are attached to the surface or embedded within a collagen gels matrix. A magnetic field that naturally has a strong magnetic field gradient pulls on the magnetic spheres that, in turn, stretch the collagen fibers enhancing the gel stiffness. Another approach is to cross-link collagen gels further using riboflavin that is known to cross-link collagen under UV light. Light masks would be employed to impose a gradient on this cross-linking. The latter two are active, ongoing projects. Our collaboration with NCI (Dalal Lab) on the various aspects of the structure/stoichiometry of the centromere made some progress looking into mutations of the centromeric cenP-A histone. The reliability of the production of the mutants came into question after several efforts to distinguish their presentation in-vivo and the lab undertook to develop in-house expertise in production of an array of mutants. The effort is ongoing. Another NCI project (Adhya Lab) made progress investigating a specific small RNA (80 bases) expressed in bacteria that strongly interact both with the DNA and with the abundant HU protein. This suggested another, hitherto unknown, pathway for nucleoid organization. We previously examined the various complexes formed between DNA and HU in the presence of the small RNA and the images pointed to definite cooperative action involving small RNA. The work expanded to a number of alternative small RNAs and additional controls hypothesizing a more general principle whereby small RNAs play a general organizational role in bacterial nucleoids. We initiated a project with NEI (Wistow Lab) to study the behavior of gamma-crystallin, a basic component in the eye lens, whose dysfunction is thought responsible for the formation of lens cataracts. The lab has a number of gamma-crystallins from different species and several mutant forms of these proteins and they are interested in the oligomerization properties of the protein under different conditions. This is to aid with the effort to crystalize these proteins for X-ray diffraction studies. Using the AFM we showed that the crystallins can form rigid fibers and small oligomers in a ring structure, depending on pretreatment, such as incubation at elevated temperatures or unfolding and refolding of the protein. This work may further clarify the factors leading to the formation of cataracts in the eye. Moreover, gamma-crystallins have also been shown to have other functions within and outside the eye. Those are of metabolic and regulatory nature. It is thought that gamma-S-crystallin binds to the actin filaments of various cell lines including the lens epithelial cells and the lens fiber cells affecting F-actin fiber stability. We are investigating the modes of that binding under physiological conditions using the imaging capabilities of the AFM. Our longstanding collaboration with NICHD (Basser Lab) continued with the study of cartilage. We are performing detailed measurements to ascertain the constitutive laws governing the mechanics of cartilage. We have been using bovine tissue measurements were performed across slices cut through the thickness of the tissue at various depths from the articular surface. In addition we helped with the visualization of the fiber formation process of a novel hydro-gelator, termed NapFFKYp. The AFM shed light to the assembly process, the dimensions of the fibers and the stiffness of the fibers thus informing on the gel formation process. This information enhances our understanding of emergent properties of hydrogels thus helping with tailoring hydrogels for a wide range of applications.